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Infect Immun. 2016 Sep 19;84(10):2995-3006. doi: 10.1128/IAI.00437-16. Print 2016 Oct.

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung.

Author information

1
Pulmonary and Critical Care Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA giffora@hitchcock.org dhogan@dartmouth.edu.
2
Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
3
Pulmonary and Critical Care Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA.
4
School of Life Sciences, Arizona State University, Tempe, Arizona, USA.
5
Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
6
Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA giffora@hitchcock.org dhogan@dartmouth.edu.

Abstract

The discovery of therapies that modulate Pseudomonas aeruginosa virulence or that can eradicate chronic P. aeruginosa lung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding of P. aeruginosa behavior in vivo We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances of P. aeruginosa transcripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiology in vitro and in vivo The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety of P. aeruginosa strains as well as RNA serial sputum samples from four P. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis of P. aeruginosa grown in vitro identified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates. P. aeruginosa transcript profiles in RNA from CF sputum indicated alginate production in vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory. P. aeruginosa gene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grown P. aeruginosa showed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CF P. aeruginosa lung infections.

PMID:
27481238
PMCID:
PMC5038062
DOI:
10.1128/IAI.00437-16
[Indexed for MEDLINE]
Free PMC Article

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